Composition for coating support for preparation of cell sheet support for preparation of cell sheet and process for producing cell sheet

ABSTRACT

Disclosed are a method for manufacturing a cell sheet, comprising culturing cells on a fibrin-coated surface of a substrate until the cells reach confluency, continuing the cultivation of the cells for a sufficient time period to cause the degradation of fibrin at the bottom of the cells, and detaching the cultured cells from the substrate surface in a sheet-like form to give a cell sheet; a substrate for cell sheet preparation, a surface of which is coated with fibrin; and a composition for use in coating with fibrin a surface of a substrate for cell sheet preparation, the composition comprising fibrinogen and thrombin. The invention enables to cell sheets to be manufactured by a simple manipulation using a substrate coated with a commercial, commonly available material of which safety has been confirmed.

FIELD OF THE INVENTION

The present invention relates to a composition for use in the coating ofa substrate for cell sheet preparation, a substrate for cell sheetpreparation, and a method for manufacturing a cell sheet.

BACKGROUND ART

In the treatment of a severely damaged heart, cell transplantationutilizing a variety of stem cells has been attempted as an alternativetherapy to heart transplantation which has been suffering from shortageof donors. Recently, based on such cell transplantation techniques,tissue transplantation techniques have been increasingly developed inwhich myocardial tissues are constructed three-dimensionally in vitroand then transplanted into a body. For example, various types of cellsheets have been successfully manufactured by usingtemperature-responsive culture dishes which are prepared by coatingpoly(N-isopropylacrylamide) (abbreviated to “PIAAm”) on the surfaces ofcommercial polystyrene culture dishes with electron beams. Inparticular, as for myocardial cells, it has already been reported thatmyocardial tissue masses available as transplants can be developed byoverlaying the thus prepared multiple myocardial cell sheets (JapanesePatent Application Laid-open No. 2003-38170, WO 01/068799 pamphlet,Simizu et al.: Fabrication of pulsatile cardiac tissue grafts using anovel 3-dimensional cell sheet manipulation technique andtemperature-responsive cell culture surface: Circ Res. 2002;90:e40-e48). The thus prepared myocardial tissue mass is found toexhibit electrical activities similar to those of normal myocardialtissues in vitro and in vivo.

With respect to primary cultures of different tissues, particularly ofmyocardial cells, there is some difference in procedure amongfacilities. The method for manufacture of cell sheets using the abovedescribed temperature-responsive culture dishes may be effective formanufacturing cell sheets with a relatively consistent efficiency if theprimary culture is performed by stringent procedures chosen for the bestmatch to such specialized culture dishes. In this method, however, it isdifficult to form sheets from cells if the procedures conventionallyemployed in each facility are applied without any modification.

Accordingly, an object of the present invention is to provide a methodfor manufacturing a cell sheet by a simple manipulation using asubstrate coated with a commercial, commonly available material of whichthe safety has been confirmed.

Another object of the present invention is to provide a composition foruse in the coating of a surface of a substrate for cell sheetpreparation.

Still another object of the present invention is to provide a substratesuitable for cell sheet manufacture.

DISCLOSURE OF THE INVENTION

The present inventors have found that, when cells are cultured forseveral days on a culture dish which was previously given a lightcoating of fibrin glue (which is degradable by most cells), the fibrinbetween the cells and the culture dish disappears and, as a result, thecells are suspended over the culture dish while binding to one anotherin a sheet-like form. Then, the inventors have established a procedurefor manufacturing cell sheets with high probability by detaching andharvesting the cell sheet intact with a scraper. These findings lead tothe completion of the present invention.

The subjects of the present invention are as follows.

(1) A composition for use in coating with fibrin a surface of asubstrate for cell sheet preparation, the composition comprisingfibrinogen and thrombin.

(2) A substrate for cell sheet preparation, a surface of which is coatedwith fibrin.

(3) A method for manufacturing a cell sheet, comprising culturing cellson a fibrin-coated surface of a substrate until the cells reachconfluency; continuing the cultivation of the cells for a sufficienttime period to cause the degradation of fibrin at the bottom of thecells; and detaching the cultured cells from the substrate surface in asheet-like form to give a cell sheet.

(4) The method according to item (3), further comprising overlaying thedetached sheet-like cultured cells.

(5) The method according to item (3) or (4), wherein the cell sheet isused in the field of regenerative medicine or in a biological activitytest of an agent.

The present invention enables cell sheets to be manufactured from avariety of cell types by employing the same procedures as those employedin various facilities without any modification, and the success rate oftheir manufacture is consistent.

The present invention also enables a large quantity of cell sheets to bemanufactured quickly using commercial fibrin glues without the need touse expensive specialized PIAAm-coated culture dishes.

This specification includes part or all of the contents as disclosed inthe specification and/or drawings of Japanese Patent Application No.2003-328340 based on which the present application claims priority.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows the generation process of a rat myocardial cell sheet.

FIG. 2 shows the generation process of a C2C12 cell sheet.

FIG. 3 shows the generation process of a mature skeletal muscle cellsheet.

FIG. 4 shows electrocardiogram data of a myocardial cell sheet.

FIG. 5 shows a rat myocardial cell sheet transplanted onto the skin.

FIG. 6 shows the result of the immunostaining of a rat myocardial cellsheet with actinin and connexin 43 two days after establishing the sheetin vitro.

FIG. 7 shows the result of the HE staining of a rat myocardial cellsheet seven days after transplantation onto a nude rat subcutaneoustissue.

FIG. 8 shows the result of the immunostaining of a rat myocardial cellsheet with actinin and connexin 43 seven days after transplantation ontoa nude rat subcutaneous tissue.

FIG. 9 shows a representative scheme of the mechanism and manipulationof a myocardial cell sheet using a polymerized fibrin-coated dish. Themanipulation was performed in the order: A→B→C→D→E→F.

Primary cultured neonate rat myocardial cells were spread onto a fibrinpolymer-coated dish (A, B, G). On day 4, the fibrin polymer was degradedby various proteases secreted from the myocardial cells (C). The cellswere gently raked from the edge toward the center of the dish so as notto tear the generated myocardial cell sheet (hereinafter, sometimesabbreviated as “MCS”) with a cell scraper (D, E), thereby obtaining ashrunken MCS (H). A few drops of a culture medium were applied onto theshrunken sheet, whereupon it was unfolded or smoothed out (E, I). Theedges of the flattened MCS were trimmed in a square shape by using ablade. In some experiments, two MCSs were overlaid on each other by themargin of 2-mm width and then co-cultured on a laminin-coated culturedish (F, J).

FIG. 10 shows the results of the histological analysis of a MCS.

(A) The protocol for preparation and histological analysis of amyocardial cell sheet. (B-E, H, I) H&E staining of a MCS. (F, G, J, K)Immunofluorescent staining of a MCS. Red: F-actin-stained cell nuclei;Green: fibrin-stained cell nuclei; Blue: TOTO-3-stained cell nuclei. Theprotocol and scale bars are indicated in the figure inset.

FIG. 11 shows the characteristic properties of MCSs.

(A) Success rate for obtaining MCSs in the PX-S0 day samples. (B)Diameter of MCSs in PX-S0 samples. (C) Percentages of spontaneousbeating in MCSs prepared using the PX-S3 day samples. (D) Percentage ofspontaneously beating sheets from the P4-SX day samples. (E) Percentagesof myocardial cell sheets that captured artificial pacing in the P4-SXday samples. (F) The beat frequency in the P4-SX day samples.

FIG. 12 shows the electrocardiogram (ECG) recorded using a pair ofcontact bipolar electrodes and the propagation of action potentialrecorded by optical mapping for analysis of electrical activities in twooverlaid MCSs.

(A) A schematic illustration of the two overlaid MCSs and the positionsof the contact bipolar electrodes. (B) A microscopic image of the twooverlaid MCSs. (C) Extracellular electrical potentials obtained from thetwo MCSs which showed synchronization in spontaneous beating. (D) Acontour map of propagation of action potential as observed by opticalmapping. The interval between each isochronal line was 35 ms. The actionpotential originated from the left lower side of sheet A, went aroundthe lower half of the junction which was an electrically unexcitablearea and propagated to sheet B via the upper localized half area of thejunction. (E) Action potential as seen to propagate by tracing theexcitation wave front (along the black curved line with arrowed head inD).

FIG. 13 is an optical mapping showing the action potential propagationof partially overlaid two MCSs one day after the co-culture of the MCSswas started.

Representative data on day 1. (A) A photographic image observed under aphase contrast microscope. (B-D) Optical images showing active potentialobtained at 14, 84, and 150 ms after pacing at the left margin of sheetA, respectively. Note that localized capture can be observed in sheet B(see the arrow) (E) A cross-sectional schematic image of the MCSs andthe site of pacing. (F) An action potential map. The interval betweeneach isochronal line is denoted 7 ms. There was crowding of theisochronal lines at the left margin of the junction, suggesting thatconduction delay started to occur around this site. Action potentialpropagation was blocked at the end of the junction. (G) Impulsepropagation sequence along the excitation wave front (see the red curvedline with an arrow in F). (H) The action potential traces at arbitrarypoints along the excitation wave front were superposed. The characterscorrespond to the positions of the myocyte from which the actionpotential was recorded.

FIG. 14 is an optical mapping showing the action potential propagationand electrical connection of overlaid MCSs on day 3.

Representative data on day 3. (A) A photographic image observed under aphase contrast microscope. (B-D) Optical mapping images showing activepotential obtained at 14, 84, and 150 ms after pacing at the left marginof sheet A, respectively. (E) A cross-sectional schematic image of theMCSs and the site of pacing. (F) Calculated activation map (the size wasthe same as that in FIG. 13F), suggesting that the propagation of actionpotential between the two MCSs was quite smooth without any conductiondelay. (G) The propagation sequence of the excitation wave frontsuggested the formation of tight electrical communication between thetwo MCSs without delay in the travel of excitation wave. (H) The actionpotential traces at arbitrary points along the excitation wave frontwere superposed.

FIG. 15 shows histological evidence for establishment of satisfactoryelectrical connection between two myocardial cell sheets in vitro.

Laser confocal microscopy of overlaid MCSs (day 3) afterimmunohistological stain. The MCSs were triple-stained with antiactininantibody (green), anticonnexin 43 antibody (red), and TOTO-3 to stainthe nucleus. (A) Top view. (B) Side view. (C) Top view observed at highmagnification. Note that the myocardial cells formed a confluent sheetand that connexin 43 was clearly observed at the cell junctions.

FIG. 16 shows the transplantation of three overlaid myocardial cellsheets in vivo.

The three overlaid MCSs were transplanted into a nude rat on thesubcutaneous tissue and the samples were observed on day 14. (A) Thetransplanted area (black dotted line) showed rhythmical spontaneousbeating (200 bpm). (B) H&E staining of the cross-sectional view of thetri-layered myocardial cell sheet graft. Sk, skeletal muscle; Ct,connective tissue; Cs, transplanted three overlaid MCSs. (C) Azanstaining of serial sections of one sample. (D) A cross-sectional view athigh magnification. Note that microvessels are apparent in thetransplanted MCSs. *Microvessels. (E) Triple staining of thetransplanted three overlaid MCSs, as described in connection with FIG.13. Note that the transplanted myocardial cells show a well-organizedsarcomere with a coincident direction of orientation.

FIG. 17 shows the result of optical microscopy (×100 magnification) ofrabbit corneal epithelial cells cultured on a fibrin sheet.

FIG. 18 shows a cultured rabbit corneal epithelial cell sheet asdetached with a scraper.

FIG. 19 shows the result of optical microscopy (×100 magnification) ofrabbit oral mucosal epithelial cells cultured on a fibrin sheet.

FIG. 20 shows a cultured rabbit oral mucosal epithelial cell sheet asdetached with a scraper.

FIG. 21 shows the result of staining keratin 3/12 in rabbit cornea andoral mucosal epithelium as positive controls.

(A) A nuclear staining image of corneal epithelium.

(B) A staining image of nuclei and keratin 3/12 of corneal epithelium.

(C) A nuclear staining image of oral mucosa.

(D) A staining image of nuclei and keratin 3/12 of oral mucosa.

FIG. 22 shows the immunostaining of cell sheets prepared using rabbitcorneal epithelial cells and oral mucosal epithelial cells.

(A) A nuclear staining image of a corneal epithelial cell sheet.

(B) A staining image of nuclei and keratin 3/12 of a corneal epithelialcell sheet.

(C) A nuclear staining image of an oral mucosal epithelial cell sheet.

(D) A staining image of nuclei and keratin 3/12 of an oral mucosalepithelial cell sheet.

In the cultured epithelium sheets, the corneal epithelium was overlaidand stained at the areas where keratin 3/12 was localized. The oralmucosal epithelium was also overlaid and the areas where keratin 3/12was localized were weakly stained. The rabbit corneal epithelium andoral mucosal epithelium cultured on fibrin sheets were overlaid and theyexpressed keratin. Accordingly, it was demonstrated that culturedepithelium having properties similar to those of normal tissues can beprepared using these sheets.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention provides a composition for use in coating withfibrin a surface of a substrate for cell sheet preparation, thecomposition comprising fibrinogen and thrombin.

Fibrin is a poorly soluble fraction produced by specific hydrolysis ofthe A alpha-chain and B beta-chain of fibrinogen by thrombin to releasefibrinopeptides A and B. The reactive residues on fibrin whichparticipate in aggregation of fibrin monomers are hydrogen-bonded to oneanother to form a fibrin polymer. The fibrin polymer can gel with acertain configuration.

Fibrinogen is a glycoprotein having a molecular weight of about 340,000,and is composed of paired sets of three types of subunits: Aalpha-chain, B beta-chain and gamma-chain having molecular weights of65,000±1,000, 55,000 and 47,000, respectively, which are bonded to oneanother through S—S bonding. The Arg-Gly bonding in fibrinogen ishydrolyzed by thrombin to release fibrinopeptides A and B from the Aalpha-chain and the B beta-chain, respectively, whereby fibrinogen isconverted into fibrin.

Thrombin is a protease which can act on fibrinogen to produce fibrin. Inthe composition of the present invention, thrombin may be present in acatalytically effective amount for converting fibrinogen into fibrin.

Fibrinogen and thrombin are preferably derived from human in view of thefact that a cell sheet prepared by cultivating cells on a fibrin coatingformed with them is intended to be used in the human body. However,fibrinogen and thrombin are not to be limited to the human origin andmay be derived from other animals such as monkey, pig, mouse, rat,baboon, canine, feline, sheep or bovine which are already on the market.

Fibrinogen and thrombin to be used in the invention may be commerciallyavailable products. For example, Tissiel Kit from Baxter can be used.Tissiel Kit contains human fibrinogen, human thrombin, calcium chloridedihydrate and aprotinin.

Preferably, the composition of the present invention further containscalcium chloride (which may be in the form of a hydrate), aprotinin,physiological saline and the like.

An example of the composition of the present invention (per 16 ml) is asfollows: fibrinogen 45 to 180 mg; thrombin 0.2 to 0.8 U; calciumchloride dihydrate 0.5 to 1.0 mg; aprotinin 1500 to 6000 U; andphysiological saline 16 ml.

In the composition, it is recommended to store fibrinogen and fibrin inseparate containers and to mix them immediately prior to use, becausethe formation of fibrin begins to occur upon the reaction of thrombinwith fibrinogen. To fibrinogen may be added serum albumin, amino aceticacid, aprotinin, tyloxapol, sodium chloride and sodium citrate. Tothrombin may be added serum albumin, amino acetic acid and sodiumchloride. It is also recommended to store calcium chloride dihydrate ina container separate from the container for fibrinogen and to mix themimmediately prior to use, because ionized calcium accelerates thehydrolysis of fibrinogen. For example, calcium chloride dihydrate may bedissolved in a solution for use in the dissolution of thrombin(hereinafter, referred to as “a dissolution solution for thrombin”)immediately prior to use and be stored in a container. Aprotinin may bepreviously added to fibrinogen or, alternatively, may be dissolved in asolution for use in the dissolution of fibrinogen (hereinafter, referredto as “a dissolution solution for fibrinogen”) immediately prior to useand be stored in a container, because aprotinin can inhibit thepolymerization of fibrinogen. Here, an example of the method of using ofthe composition will be described. First, fibrinogen is dissolved in adissolution solution for fibrinogen containing aprotinin to preparesolution A. Then, thrombin is dissolved in a dissolution solution forthrombin containing calcium chloride dihydrate to prepare solution B.Solutions A and B and physiological saline are mixed together and themixed solution is applied onto a surface of a substrate for cell sheetpreparation.

The composition of the present invention can be used to coat a surfaceof a substrate for cell sheet preparation with fibrin.

Accordingly, the present invention provides a substrate for cell sheetpreparation, a surface of the substrate being coated with fibrin.

The substrate may be of any type, as long as cells can be cultured onit. Examples of the substrate include a culture dish, a Petri dish, aculture plate having 6 to 96 wells, and Celldex LF (SUMILON). Thematerial for the substrate may be exemplified by, but is not limited to,glass, modified glass, polystyrene, polymethyl methacrylate, andceramics.

For manufacture of the substrate for use in the cell sheet preparation,the composition of the present invention may be applied onto a surfaceof the substrate to form a fibrin coating thereon. An example of theprocedure will be described in detail below.

Fibrinogen, thrombin, calcium chloride dihydrate, aprotinin andphysiological saline are mixed together and the mixed solution is thenapplied onto a surface of the substrate. The substrate is then allowedto stand for an appropriate time period (usually 1 to 3 hours) at roomtemperature to form fibrin thereon. The resulting substrate may bestored under sterile conditions at 4° C. until it is used as a substratefor cell sheet preparation.

The present invention provides a method for manufacturing a cell sheet,comprising culturing cells on a fibrin-coated surface of a substrateuntil the cells reach confluency; continuing the cultivation of thecells for a sufficient time period to cause the degradation of fibrin atthe bottom of the cells; and detaching the cultured cells from thesubstrate surface in a sheet-like form to give a cell sheet. A film-likesheet can be obtained by raking the cells which have been grown on aculture dish densely until they reach confluency. As used herein, theterm “confluency” means the state where cells are placed densely withoutleaving any gap and it can be observed under a microscope.

Examples of the cell to be cultured include, but are not limited to,myocardial cell, skeletal myoblast, mature skeletal muscle cell, smoothmuscle cell, bone marrow stromal cell, corneal epithelial cell, oralmucosal epithelial cell and dermal cell. For the cultivation of thecells on a culture dish to confluency, there are two approaches: oneapproach is by spreading cells of a single type; and the other approachis by spreading multiple types of cells simultaneously. For thecultivation of a single type of cells to confluency, there are twoapproaches: one approach is to plate a small amount of monoclonal cellshaving proliferation potency on a culture dish and then grow the cellsuntil they reach confluency; the other approach is to plate a largeamount of polyclonal cells having poor proliferation potency on aculture dish and, when they adhere onto the bottom of the culture dish,grow the cells until they reach confluency. As one example of the formerapproach, cells of an immortalized cell line (e.g., C2C12 strain cellsderived from murine skeletal myoblasts, CMG cells, etc.) are plated in asmall amount and grown on a culture plate until the cells reachconfluency. As one example of the latter approach, myocardial cells,skeletal myoblasts, bone marrow stromal cells and the like are harvestedfrom cardiac muscle, skeletal muscle, smooth muscle, bone marrow and thelike, respectively, by primary culture techniques, the cells areselectively collected by means of a cell sorter, percoll oradhesion-based separation technique to increase the cell purity, andthen a sufficient amount of the cells are plated on a culture dish. Asone example of the approach for growing multiple types of cells toconfluency, fibroblasts are mixed to myocardial or skeletal muscle cellsbefore a cell sheet is formed from them. In this case, even if thenumber of the myocardial or skeletal muscle cells used is insufficient,fibroblasts which have high proliferation potency invade into the gapsamong the myocardial or skeletal muscle cells and the entire bottomsurface of the culture dish is covered with either type of cells, thusachieving a confluent state. In this manner, even cells which aredifficult to harvest in a sufficient amount and which have poorproliferation potency can be grown to permit easy formation of a cellsheet by co-cultivation of “bridge-cells” such as fibroblasts. As suchbridge-cells, not only fibroblasts but also smooth muscle cells andendothelial cells may be used. Depending on the type of cells used as“bridge”, the strength and stretching property of a cell sheet can bemodified for intended use.

The cells may be derived from human and non-human animals (e.g., monkey,pig, mouse, rat, baboon, canine, feline, sheep or bovine). The cells maybe harvested directly from the source such as an animal or they may becultured cells of an established or unestablished cell line.

The manufacture of a cell sheet can be achieved by culturing cells onthe fibrin-coated surface of a substrate until the cells reachconfluency; continuing the cultivation of the cells for a sufficienttime period to cause the degradation of fibrin at the bottom of thecells; and detaching the cultured cells from the substrate surface in asheet-like form to give a cell sheet. The cultivation of the cells maybe conducted by any method or under any condition as long as thecultivation is conducted on the fibrin-coated surface of a substrate.The cultivation may be continued until the cells reach confluency andthe fibrin is degraded to the extent that the cells can be detached fromthe substrate surface in a sheet-like form. If it is required to culturethe cells for a prolonged period of time before sheet formation, anappropriate amount of aprotinin may be added to the culture several daysbefore sheet manipulation. In this manner, the cultivation can beprolonged by a desired number of days without causing degradation offibrin. Generally, the cells are cultured in a culture medium until theybecome confluent, and the cultivation is continued for an additionalthree to four days in a culture medium without aprotinin. During thecultivation, degradation of the fibrin at the bottom of the cells occursspontaneously due to the cultured cells. In the cultivation, a substancecapable of degrading fibrin (e.g., plasmin) may be added to the culturemedium to intentionally control the degradation rate of fibrin.Thereafter, the culture medium may be aspirated off and the resultingcell sheet may be detached from the substrate in a film-like form usingdetaching means such as a scraper. After the cell sheet is detached, afew drops of a fresh culture medium may be applied onto the cell sheetto unfold or smooth out the sheet.

The cultured cells detached in a sheet-like form may be overlaid to forma multiple layers. An example of the procedure for overlaying the cellsheets is described below.

From a first cell sheet which has been unfolded on a culture dish, theculture medium is further aspirated off, and the cell sheet is allowedto stand in a saturated steam incubator at 37° C. for an appropriatetime (e.g., 15-30 min.). During this time period, the first cell sheetadheres to the culture dish. A second cell sheet as just detached from aculture dish is aspirated along with the culture medium by means of apipette and then applied onto the first cell sheet fixed on the culturedish. A few drops of the culture medium are gently applied onto thesecond cell sheet placed in a shrunken state on the unfolded first cellsheet, whereby the second cell sheet can be unfolded while beingoverlaid on the first cell sheet. The same procedure is repeated tooverlay one cell sheet on another.

By using the method of the present invention to form a cell sheet ofvarious cell types or to overlay cell sheets, tissue grafts for avariety of organs can be generated in vitro. Use of the tissue graftsthus prepared enables the establishment of analytical procedures invitro at the cellular to tissual level.

The cell sheets manufactured by the method of the present invention canbe used in the field of regenerative medicine or in biological activitystudy on an agent.

As the cell sheets for use in regenerative medicine, there may bementioned a myocardial cell sheet, a corneal epithelial cell sheet, anoral mucosal epithelial cell sheet, a dermal cell sheet and the like. Amyocardial cell sheet can be used for treatment of heart failure andarrhythmia resulting from cardiac infarction and various types ofmyocarditis and cardiomyopathy and as a material for cardiac muscletransplantation. A corneal epithelial cell sheet and an oral mucosalepithelial cell sheet can be used as materials for keratoplasty. Adermal cell sheet can be used for the treatment of wounds resulting fromburns and injuries and the like. It may also be possible to use afibroblast cell sheet in therapy for wound cure promotion.

The biological activity test of an agent may be exemplified bypharmacological activity test, toxicity test and biding activity test ofan agent. Examples of the binding activity of an agent includeligand-receptor binding activity and antibody-antigen binding activity.In comparison with the conventional methods for examining the change incell behavior that results from addition of various agents to a culturemedium for cell cultivation, the addition of such various agents to acell sheet culture medium to examine the effect on the cell sheetenables examining not only the effect on cells themselves but also theeffect on intercellular structure and construction. It is also possibleto examine such effects of an agent at the cellular level, as well as atthe organ level. Cell sheets derived from different human organs can betransplanted onto organs of immunodeficient animals (e.g., nude mice,skid mice, nude rats) and, after administration of an agent to thetransplantation model animals, the state of the cell sheets can beexamined to predict the effect of the agent on human organs in vivo.

By the biological activity tests of agents using the cell sheetmanufactured by the method of the present invention, candidatesubstances for medicines and agricultural chemicals having desiredbiological activities can be screened.

The present invention will be described in great detail with referenceto the following examples. However, it should be understood that theseexamples are for illustrative purposes only and that the scope of theinvention is not limited thereto.

The commercial suppliers and preparation methods of the materials usedin the examples are as follows:

Culture dishes (FALCON 35 3001, diameter=3.5 cm);

Wistar rats (Japan CLEA);

2.5% Trypsin (GIBCO 15090-046);

Collagenase (SIGMA C-5138);

DNase I (SIGMA DN-25);

FBS (JRH Bioscience);

Penicillin, streptomycin, amphotericin B (GIBCO 15240-062);

Medium 199 (ICN Biomedicals 1023126);

DMEM (GIBCO 12100-046);

C2C12 derived from murine skeletal myoblasts (purchased from ATCC);

CMG cells (a cell line produced by cloning an immortalized line ofmurine bone marrow cells that acquired the ability to be transformed tocardiac cells by treatment with 5-azacytidine in CardiopulmonaryDivision, Department of Internal Medicine, Keio University School ofMedicine);

Nude rats (Japan CLEA);

Rabbit anti-mouse connexin antibody (SIGMA C6219);

Mouse anti-actinin antibody (SIGMA A7811);

TRITC-conjugated pig anti-rabbit IgG antibody (DAKO R 0156);

Alexa488-conjugated goat mouse IgG antibody (Molecular Probes A-11029);

Rabbits (Japanese white rabbits, female, about 3 kg, Shiraishi LaboratyAnimal Care Company (Shiraishi Jikken-doubutsu Shiikujo));

SHEM

DMEM/F12: Gibco BRL D-MEM/F12 (1 pack/for 1 L, 12400-016; 15.6 g/unit;containing HEPES);

NaHCO₃: Waco (concentration in use: 2.5 g/l);

Insulin: SIGMA human recombinant expressed in E. coli; I-0259; 50mg/unit (concentration in use: 5 μg/ml));

Human-EGF: Gibco BRL Recombinant Human EGF; 13247-010; 100 μg/unit(concentration in use: 10 μg/ml);

Cholera toxin: SIGMA c-2012; 1 mg/unit (concentration in use: 1 μg/ml);

0.5% DMSO: SIGMA DIMETHYL SULFOXIPE; D-2650; 5 ml×5 tubes/unit);

15% FCS: Sanko Junyaku (Vitromex); VMS 1500; Lot, F000210802; 500 mlDMEM (Gibco);

Fetal bovine serum (Nichirei);

3T3 cells (American Type Culture Collection);

Aprotinin (Wako);

Gentamicin, penicillin, anphotericin B (Wako);

Dispase II (Godo Shusei);

DMEM/F12 (Gibco);

Trypsin (Gibco);

EDTA (Gibco);

Normal Donkey Serum (CHEMICON; S30-100ML; Lot, 23031387; 100 ml);

BSA (Sigma);

DAPI (Sigma).

EXAMPLE 1 Coating of Culture Dish with Human Fibrin

Human fibrinogen (90 mg), thrombin (0.4 U), calcium chloride dihydrate(0.59 mg) and aprotinin (3000 U) (these components were used as Tissiel1-ML Kit (Baxter)) and physiological saline (16 ml) were mixed togetherand quickly spread onto the bottom of a 35-cm culture dish (0.3ml/dish). The dish was allowed to stand for 1 hour at room temperaturewhile it was kept level. Thereafter, the culture dish on which fibrinhad formed and the Tissiel dilution solution had solidified at thebottom was stored at 4° C. while it was kept sterile. Stored in thisstate, the culture dish is effective for use for about 2 months. Fortyto fifty 3.5-cm culture dishes could be prepared using the Tissiel 1-MLKit.

EXAMPLE 2 Cell Culture on Fibrin-Coated Culture Dishes

(1) Rat Myocardial Cells

The ventricles were removed from 1 day-old neonatal Wister rats andenzymatically treated with 0.03% trypsin, 0.03% collagenase and 20 μg/mlDNase I to isolate ventricular myocytes. Two milliliters of medium199/DMEM supplemented with 10% FBS and penicillin (50 U/ml)/streptomycin (50 μg/ml) /amphotericin B (25 μg/ml) and the cells (2×10⁶cells) were injected into each of the fibrin-coated 3.5-cm culturedishes, and the cells were cultured in a 5% CO₂ incubator at 37° C.

(2) C2C12 Cells Derived from Murine Skeletal Myoblasts

Cell line C2C12 derived from murine skeletal myoblasts purchased fromATCC was cultured using DMEM culture medium supplemented with 10% FBS ina 5% CO₂ incubator at 37° C. and passaged at 80% confluency.

(3) Mature Skeletal Muscle-Like Cells Derived from Murine C2C12 Cells

The C2C12 cells (1×10⁷ cells) which had been passaged according to theprocedure (2) were seeded in a 75-cm flask and DMEM (20 ml) supplementedwith 5% horse serum was added. The culture medium was replaced by afresh one at a frequency of once or twice a week while checking thestate of the cells.

(4) Bone Marrow Stromal Cells

The bone marrow was removed under sterile conditions from the thigh boneof mice. The nucleated cells were plated on fibrin-coated 3.5-cm culturedishes at a density of 1×10⁷ cells/dish and 3 ml of a mesenchymal stemcell growth medium (PT-3001) (Sanko Junyaku) was added. The medium wasreplaced by a fresh one at a frequency of once a week.

EXAMPLE 3 Generation of Cell Sheets from Cells

(1) Preparation of Rat Myocardial Cell Sheets

Four days after the primary culture, the cells were beatingspontaneously at the bottom of the culture dish in a confluent state.The culture medium was aspirated off and the myocardial cells which hadbeen bonded to one another in a sheet-like form were gently raked with ascraper from the edge toward the center of the culture dish so as nottear the myocardial cell sheet. After the cell sheet was completelydetached, a few drops of a fresh culture medium were applied to theshrunken cell sheet carefully to unfold the cell sheet on the culturedish. The generation of the cell sheet from rat myocardial cells isshown in FIG. 1.

(2) Preparation of Cells Sheets from C2C12 Cells, Mature SkeletalMyoblasts and Bone Marrow Stromal Cells

According to the same procedure as in Example 2, cell culture wascontinued until the cells reached confluency on a fibrin-coated culturedish. After continuing the cultivation for an additional 3 to 4 days, acell sheet in a film-like form was detached with a scraper in the samemanner. The generation process of the cell sheets from C2C12 cells andmature skeletal myoblasts are shown in FIGS. 2 and 3, respectively.

EXAMPLE 4 Multiple Lamination of Cell Sheets

A first cell sheet was unfolded on a culture dish by applying a fewdrops of a culture medium onto the sheet as described in Example 3. Theculture medium was aspirated off from the cell sheet as much aspossible, and the cell sheet was then allowed to stand in a saturatedsteam incubator at 37° C. for 15 min. During this time period, the firstcell sheet adhered to the culture dish with a weak force. Next, a secondcell sheet as just detached from a culture dish was aspirated along witha culture medium by means of a 10-ml pipette and applied onto the firstcell sheet which had been fixed on the culture dish. A few drops of afresh culture medium were carefully applied onto the second cell sheetplaced in a shrunken state on the unfolded first cell sheet, whereby thesecond cell sheet was unfolded as it was overlaid on the first cellsheet. The same procedure was repeated to overlay multiple cell sheetssuccessively.

EXAMPLE 5 Functional Analysis of Myocardial Cell Sheets

(1) Electrical Activities In Vitro

To two partially overlaid myocardial cell sheets was added 1 ml of aculture medium. Culture of the cell sheets was continued while theculture medium was replaced daily with a fresh one. After two days, thetwo cell sheets were observed under a microscope and they exhibitedspontaneous contraction at the same beating rate. The electrocardiogrammeasured at the both edges of each myocardial cell sheet revealed thatthe two cell sheets were contracted in the same rhythm (FIG. 4).

(2) In Vivo Take

Two overlaid rat myocardial cell sheets were transplanted into 5week-old nude rats on the subcutaneous tissue. One week after thetransplantation, the skin was incised and the myocardial cell sheetswere observed (FIG. 5). It was confirmed with the naked eye that thecell sheets showed rhythmical contraction.

(3) Study of Muscle Contractile Protein and Gap Junction byImmunohistological Staining

Two days after in vitro preparation of a rat myocardial cell sheet,actinin (a representative contractile protein found in myocardial cells)and connexin 43 (a constitutive protein of gap junction) wereimmunostained. The result is shown in FIG. 6. Two overlaid ratmyocardial cell sheets were transplanted into a 3 week-old nude rat onthe subcutaneous tissue in vivo. Seven days later, the cell sheets wereremoved and subjected to HE staining and immunostaining of actinin andconnexin 43. The results are shown in FIGS. 7 and 8, respectively. Itwas demonstrated that the expression of actinin and connexin 43 wasmaintained satisfactorily both in vivo and in vitro. In the in vivomodels, fiber orientation in myocardial cells was more marked than thein vitro models. From the HE staining, it was observed that microvesselsinvaded into the gaps between the transplanted myocardial cell sheets tosupply bloodstream.

EXAMPLE 6 Preparation, Transplantation, Histological Analysis andElectrical Activity Analysis of Myocardial Cell Sheet

Materials and Method

Preparation of Myocardial Cell Sheet

Tissiel (including human fibrinogen, thrombin, calcium chloride andaprotinin) was purchased from Baxter. Human fibrinogen (90 mg), humanalbumin (20 mg), thrombin (0.4 U), calcium chloride dihydrate (0.59 mg)and aprotinin (3000 U) were diluted with physiological saline (15 ml),and a portion (0.3 ml) of the solution was spread onto a 35-mm culturedish. About 2 hours later, a culture dish of which a surface was coatedwith fibrin polymer was obtained. This culture dish can be stored understerile conditions at 4° C. for about one month. According to the sameprocedure described above, myocardial cells could be prepared fromventricular muscle of 1 day-old neonatal Wister rats (Kodama H., FukudaK., Pan J. et al., Leukemia inhibitory factor, a potent cardiachypertrophic cytokine, activates the JAK/STAT pathway in ratcardiomyocytes. Circ Res. 1997; 81:656-663). The obtained myocardialcells were plated on a fibrin-coated culture dish at a density of2.8×10⁵ cells/cm² (FIGS. 9A, B, G). The polymerized fibrin was graduallydegraded by non-specific proteases secreted from the cultured cells. Inthree to seven days after the cultivation was started, the contactbetween the cells and the surface of the culture dish gradually becamesparse (FIG. 9C).

Thus, on day 4, the myocardial cells could be detached from the surfaceof the culture dish with a cell scraper to give a myocardial cell sheet(FIGS. 9D, H). The shrunken myocardial cell sheet was unfolded by beingsuspended in a culture medium (FIGS. 9E, I), and then trimmed in asquare shape. Two myocardial cell sheets were partially overlaid (FIGS.9F, J) for subsequent analytical experiments, and co-culture wascontinued on a laminin-coated culture dish for 1 to 3 days according tothe procedure described previously (Murata M., Fukuda K., Ishida H. etal., Leukemia inhibitory factor, a potent cardiac hypertrophic cytokine,enhances L-type Ca²⁺ current and [Ca²⁺]i transient in cardiomyocytes. JMol Cell Cardiol., 1999; 31:237-245).

Transplantation of Myocardial Cell Sheet Graft Onto Adult RatSubcutaneous Tissue

All experimental procedures were approved by the Animal Care and UseCommittee of Keio University and conformed to the NIH Guide for the Careand Use of Laboratory Animals. After inhalation of diethyl ether, maleF344 nude rats (8-week old, n=10) were topically injected on the dorsalskin with 1% procaine hydrochloride (5-10 ml) and the dorsal skin wasincised. Three overlaid myocardial cell sheets were transplanted ontothis area.

Histological Analysis

Immunohistological staining was performed as described previously(Agbulut O., Menot M L., Li Z. et al., Temporal patterns of bone marrowcell differentiation following transplantation in doxorubicin-inducedcardiomyopathy, Cardiovasc Res. 2003; 58:451-459) by using anti-fibrinantibody (Monosan, the Netherlands), anti-alpha-actinin antibody (Sigma)and connexin 43 antibody (Sigma). The samples were subjected tosecondary staining with either Alexa488-labelled anti-mouse IgG antibody(Molecular Probes) or TRITC-labeled anti-rabbit IgG antibody (Dako). Insome experiments, staining with Alexa594-labeled phalloidin (MolecularProbes) was also performed. Nuclei were stained with TOTO-3 (Sigma) Thestained samples were observed under a confocal laser microscope (LSM510,Zeiss).

Analysis of Myocardial Cell Sheet on Electrical Activities by OpticalMapping System

Two myocardial cell sheets were overlaid by the margin with 2-mm width.Extracellular electrical potentials were measured at both ends of eachmyocardial cell sheet with a pair of bipolar electrodes.

The optical mapping system was applied by using a membrane voltageresponsive dye, di-4-ANEPPS (Molecular Probes), to recordtwo-dimensional action potential propagation and evaluate the actionpotential propagation between the two overlaid myocardial cell sheets.

Di-4-ANEPPS stock solution (20 mM) was dissolved in DMSO containing 20%pluronic F-127 (P-3000, Molecular Probes), and diluted with the culturemedium to a final concentration of 10 μM di-4-ANEPPS. The samples wereallowed to stand in an incubator at 37° C. for 30 min. Thereafter, theculture medium was replaced by Tyrode's solution consisting of (mmol/l)140 NaCl, 4 KCl, 0.5 MgCl₂, 1.8 CaCl₂, 5 HEPES, 55 D-glucose (pHadjusted to 7.4 with NaOH), and 100 mg/l BSA. The culture dish havingthe myocardial cell sheets thereon was set in a temperature-controlledperfusion apparatus (37° C.) and then placed on the stage of afluorescence microscope (BX50WI, Olympus, Japan). A high-resolution CCDcamera system (MiCAM01, Brain Vision, 192×128 points, 3.5 msec timeresolution) was used to record signals from the samples at an emissionwavelength of 610 nm or longer and an excitation wavelength of 520 nm.The myocardial cell sheet was immobilized using cytochalasin-D (25 μM).Action potentials were recorded under spontaneous beating or pacingstimulation by a bipolar silver chloride electrode. The obtained datawas processed with our original analysis program produced using Igor Prosoftware (Wavemetrics) according to a method described previously (KouraT., Hara M., Takeuchi S. et al., Anisotropic conduction properties incanine atria analyzed by high-resolution optical mapping: preferentialdirection of conduction block changes from longitudinal to transversewith increasing age. Circulation. 2002; 105:2092-2098).

Results

Histological Analysis of Myocardial Cell Sheets Prepared UsingFibrin-Coated Culture Dish

For the analysis of myocardial cell sheets prepared by the presentmethod, the inventors prepared cell sheets using a cell scraper atdifferent time points after plating of myocardial cells on fibrin-coatedculture dishes (FIG. 10). The myocardial cell sheets could be detachedfrom the culture dishes after three days onward. The detached myocardialcell sheets decreased in diameter by 38±3.6% (n=30) as compared with thediameter of the culture dishes. Attempts were also made to culturemyocardial cells on non-coated, gelatin-, laminin-, orfibronectin-coated culture dishes to confluency and then detach theproduced cell sheets with a cell scraper according to the sameprocedure. However, in those cases, it was impossible to harvest thecells in a sheet form (data not shown). In the experiment underconsideration, the inventors defined the time interval (days) betweenthe primary culture and the myocardial cell sheet manipulation as “PXdays”, and the time interval (days) between the manipulation of cellsheets and the data recording as “SX days”. In a myocardial cell sheetmanipulated four days after the primary culture (P4-S0), residual fibrinwas observed at the bottom of the sheet. However, in a myocardial cellsheet manipulated six days after the primary culture (P6-S0), noresidual fibrin was observed.

In a P4-S1 myocardial cell sheet, residual fibrin was still observedbetween the myocardial cell sheet and the culture dish. However, in aP4-S3 myocardial cell sheet which was obtained after continuing thecultivation for an additional two days, fibrin completely disappeared.Interestingly, when 600 KIU/ml of aprotinin was added to the culturemedium, a considerable amount of fibrin was observed to have remainedundigested between the cells sheet and the culture dish (H-K). Theseexperimental results suggest that six days is necessary to completelydigest residual fibrin between a myocardial cell sheet and a culturedish.

Characteristic Properties of Myocardial Cell Sheets Prepared UsingFibrin-Coated Culture Dishes

First, the optimal time interval (days) between the initiation ofprimary culture and the cell sheet manipulation was determined (FIG.11). The success rate for obtaining myocardial cell sheets began toincrease after three days and peaked on day 4 (success rate=100%, n=12each) (FIG. 11A). The diameter of the obtained myocardial cell sheetsgradually increased according to the time interval between the primaryculture and the sheet manipulation due to the increase in cell densityor the mechanism of cell stretching (FIG. 11B). Next, the percentage ofspontaneously beating myocardial cell sheets among the myocardial cellsheets manipulated at different time points after the primary culturewas determined following three days of continued cultivation aftermanipulation the sheets. The time interval (PX days) between the primaryculture and the cell sheet manipulation did not affect the percentage ofresumption of spontaneous beating of the myocardial cell sheets.However, when the occurrence of spontaneous beating was observed underthe conditions where the timing of the manipulation of the myocardialcell sheets was fixed to day 4 after the primary culture (P4) andculture of the cell sheets was continued after the sheet manipulation,the percentage of spontaneous beating began to increase significantlyafter two days and 100% of the myocardial cell sheets exhibitedspontaneous beating on day 6 (S6) (6 days SX; FIGS. 11C, D). Further, weexamined whether the myocardial cell sheets would respond to pacingstimulation to exhibit rhythmic contraction, and it was found that thepercentage of myocardial cell sheets that exhibited rhythmic contractionbegan to increase on day S2 and reached 100% on day S5 (FIG. 11E). FIG.11F shows the time course of beat frequency (beating rate) of myocardialcell sheets that exhibited spontaneous contraction after theirmanipulation. Based on these results, it was decided to use a P4-S3myocardial cell sheet for the subsequent electrophysiological analysis.

Comparison of Optical Mapping System with the Approach of Using BipolarElectrodes for Analysis of Action Potential Propagation

We examined the action potential propagation within a myocardial cellsheet and determined whether action potential would propagate betweentwo myocardial cell sheets via junctions. The data of recordingelectrocardiogram on myocardial cell sheets as measured by contact withbipolar electrodes was compared with the data of recording actionpotential as measured by an optical mapping system (FIG. 12). In theelectrocardiogram recordings, it was observed that sheets A and Bexhibited synchronous spontaneous beating. In the electrocardiogram, theQRS complexes in sheets A and B were completely synchronous, suggestingthat the two myocardial cell sheets had established an electricalconnection. In the recordings of active potential by the optical mappingsystem, however, it was unexpectedly observed that the action potentialarose from the lower left side of sheet A toward the upper right sideand propagated to sheet B via the upper junction and spread throughsheet B. This observation demonstrates that the propagation of actionpotential does not follow a direct route at the junction between the twosheets. It was shown that the conduction velocity for action potentialat the area indicated by the arrow in FIG. 12D was satisfactory withoutcausing any delay or disturbance (FIG. 12E). Thus, it was found that theoptical mapping system is the sole useful technique for analyzing theaction potential propagation within a myocardial cell sheet and theelectrical connection between two myocardial cell sheets.

Constitution Process of Electrical Connection Between Two MyocardialCell Sheets

To investigate how electrical connection was constituted between twomyocardial cell sheets, the inventors analyzed the propagation of actionpotential in partially overlaid two myocardial cell sheets by theoptical mapping system. In P4-S1 myocardial cell sheets, the actionpotential did not propagate from one miocardial cell sheet to the other(FIG. 13). Interestingly, in some cases ( 3/10), partial capture ofaction potential was observed in sheet B (the arrows in FIG. 13D). FIG.13F is a contour map showing the propagation of action potential bymeans of isochronal lines. The conduction velocity of the excitationwave front on each line is shown in FIG. 13G, and it was revealed thatpropagation of action potential was blocked at site t.

These experimental results suggest that electrical connection is locallyestablished between two P4-S1 myocardial cell sheets but stableelectrical connection is yet to be established between the two sheets.

In contrast, in P4-S3 myocardial cell sheets, action potentialpropagated from sheet A to sheet B without conduction delay (FIG. 14).In all of the test samples, satisfactory electrical connection wasestablished between two myocardial cell sheets (n=10/10), and noconduction delay was observed in the conduction velocity analysis. Theseexperimental results suggest that a co-culture period three days (S3) orlonger is necessary to form stable electrical connection betweenoverlaid myocardial cell sheets.

Histological Study of Attachment Between Two Myocardial Cell Sheets (InVitro Model)

An immunofluorescent staining image of P4-S3 myocardial cell sheet isshown in FIG. 15. Sarcomeres are clearly visible in the myocardialcells. Cx43 was localized at the junctions of the myocardial cells. Thesarcomeres seemed to have such a tendency that they were oriented in thecoincident direction upon mutual contact of myocardial cells. The twooverlaid myocardial cell sheets were approximately 15±2 μm thick. Thetwo cell sheets were completely connected and it was impossible torecognize their boundary.

In Vivo Transplantation Model and Histological Study of Three OverlaidMyocardial Cell Sheets

Three overlaid myocardial cell sheets were transplanted together intonude rats on the subcutaneous tissue and observed after 14 days. Thecell sheet showed strong periodic contraction (FIG. 16). The HE- andAzan-staining revealed that the transplanted myocardial cell sheets weresandwiched between host-derived connective tissues (FIGS. 16B, C). Themyocardial cell sheets were 102±11 μm thick, thicker than when the invitro cultivation of three overlaid myocardial cell sheets wascontinued. Confocal laser microscopy showed that the size of eachmyocardial cell in vivo was greater than that of the myocardial cells inmyocardial cell sheets prepared in vitro. In addition, richneovascularization was observed in the transplanted myocardial cellsheets (FIG. 16D). This vasculature had diameters in the range of 10 to25 μm, which is not at the capillary level but at the microvessel level.The sarcomere in the myocardial cells was well organized and thedistribution of myocardial cells had such a tendency that they wereoriented in the coincident direction (FIG. 16E). These findings indicatethat myocardial cell sheets obtained using fibrin-coated culture dishesremained functional in the tissues, had tissue structures similar tothose of the normal heart and had good contraction potency.

Discussion

Since methods for preparing cell sheets using PIAAm-coated responsiveculture dishes were reported, cell sheet engineering has been advancedas one of the approaches of two- or three-dimensional tissue engineeringin a variety of organs. As a consequence, cell sheet engineering isbeginning to assume a more dominant position in the field ofregenerative medicine now. Hitherto, preparation of cell sheets fromvascular endothelial cells, hepatocytes, renal endothelial cells,corneal epithelial cells and the like using temperature-responsiveculture dishes has been reported (Shimizu T., Yamato M., Kikuchi A. etal., Cell sheet engineering for myocardial tissue reconstruction.Biomaterials. 2003; 24:2309-2316).

The method reported herein by the present inventors has severaladvantageous characteristics. First, cell sheets can be prepared easilymerely by using readily available fibrin without the need of specializedfacilities. Accordingly, the method enables cell sheets of optimal sizeand various shapes to be prepared without the need of specializedtechniques. Second, cell sheets can be prepared from any type ofadhesive cells. This is because that even cells that find difficultyadhering to conventional uncoated cell culture dishes orfibronectin-coated culture dishes exhibit extremely good adhesiveproperties onto fibrin-coated culture dishes. Third, cell sheets can beharvested rapidly. At the time when the present method was developed,there was a fear that the cellular structure of myocardial cell sheetsmight be impaired during the detachment of the cells in a sheet-likeform with a cell scraper. However, there was no visible cell necrosisunder microscopic observation. The reasons for this result are assumedas follows: the attachment of myocardial cells to the culture dishes isgradually loosened as fibrin is steadily degraded by non-specificproteases secreted from the myocardial cells; and the trace amount ofresidual fibrin may serve a cushion-like function to protect the cellsagainst physical damage during harvest. The present inventors haveconfirmed that the residual fibrin completely disappears within a totalof seven days after the primary culture of the myocardial cells.However, the length of this period may vary depending on the nature ofproteases secreted from the cells themselves, cell density at the pointof time when the cells reach confluency, and the like.

As an exemplary enzyme that is secreted from the liver in vivo and whichhas potent proteolytic effect, plasmin is well known (Ritchie D G, LevyB A, Adams M A et al. Regulation of fibrinogen synthesis byplasmin-derived fragments of fibrinogen and fibrin: an indirect feedbackpathway. Proc Natl Acad Sci USA 1982; 79:1530-1534). Even if fibrinremains in myocardial cell sheets at the point in time when the cellsheets are detached from the culture dishes, the residual fibrin isbelieved to disappear by the action of endogenous plasmin aftertransplantation. Based on the results shown herein, the method forpreparing various types of cell sheets using fibrin-coated culturedishes is considered as a practical and simple procedure in myocardialcell sheet engineering.

In order that myocardial cell sheets can be used as transplantationgrafts useful in the field of myocardial tissue engineering, goodpropagation and communication of action potential within or between themyocardial cell sheets are required. For this purpose, the opticalmapping system is believed to be an extremely effective means fordetailed examination of action potential propagation. In two overlaidmyocardial cell sheets which had been confirmed to exhibit completelysynchronized contraction under microscopic observation, analysis by theoptical mapping system revealed that active potential propagated in sucha way as to go around the junctions via areas where good electricalconnection was established. As a consequence, electrophysiologicalstudies by the optical mapping system are considered to be extremelyeffective for analyzing the presence of electrical connection between ahost and a graft after the transplantation of the graft onto the heartof the host. According to the experiments shown herein, it suggestedthat three days is necessary to establish satisfactory electricalconnection between two myocardial cell sheets. Experiments for studyingthe process of electrical connection establishment in myocardial cellsheet-transplanted models using the optical mapping system are alsounderway.

In conclusion, it is considered that the method for preparing myocardialcell sheets and the method for electrophysiological analysis ofmyocardial cell sheets, as disclosed herein, will largely contribute toadvances in the field of regenerative medicine for heart and otherorgans.

EXAMPLE 6 Generation of Cell Sheets from Cells

(1) Preparation of Corneal Epithelial Cell Sheet

The following procedure was taken to prepare a corneal epithelial cellsheet on a fibrin sheet that had been applied to a surface of a tissueculture dish (IWAKI).

1) The Descemet's membrane and iris were scraped from a rabbit with aswab and the cornea was removed therefrom.

2) The resulting sample was split into 12 pieces and placed on thefibrin-coated inner well with the epithelium side down.

3) SHEM (0.6 ml) supplemented with aprotinin (666 KIU/ml) was added tothe inner well. When the epithelium began to grown, the amount ofaddition of SHEM (supplemented with 666 KIU/ml of aprotinin) was changedto 1 ml.

4) DMEM+FCS (+), a culture medium used for cultivation of 3T3 cells, wasreplaced by 2 ml of SHEM (supplemented with 666 KIU/ml of aprotinin).

5) When the epithelium reached confluency (which usually requires 1 to 2weeks), air lift was performed (for about 1 week) The culture mediumused is SHEM without aprotinin.

6) The cultured epithelium was detached with a scraper.

A photographic image (×100) of the rabbit corneal epithelial cells grownon the fibrin sheet observed under an optical microscope is shown inFIG. 17. A photographic image of the cultured epithelium sheet detachedwith a scraper is shown in FIG. 18. Using rabbit corneal epithelialcells cultured on a fibrin sheet, a thick epithelium sheet could beprepared by means of co-cultivation of 3T3 cells and air lifting.

(2) Preparation of Oral Mucosal Epithelial Cell Sheet

The following procedure was taken to prepare an oral mucosal epithelialcell sheet on a fibrin sheet that had been applied to a tissue culturedish (IWAKI).

1) The oral mucosa was collected from a rabbit (2 mm×2 mm×1 mm specimensfrom two sites).

2) The tissue was washed (three times with a medium supplemented withgentamicin and antibiotics/antifungal agents at RT, each lasting for 15min, then once with antibiotics-loaded PBS(−) at RT for 15 min).

3) The sample was treated with Dispase II (at 37° C. for 1 hour).

4) The epithelium was removed from the parenchyma and placed in DMEM/F12+FCS (−).

5) The sample was treated with trypsin-EDTA (at RT for 8 min).

6) The sample was centrifuged at 1500 rpm for 5 min [then plated on aninner well at a density of 2×10⁵ cells/ml SHEM (supplemented with 666KIU/ml of aprotinin)].

7) DMEM+FCS (+), a culture medium used for cultivation of 3T3 cells, wasreplaced by 2 ml of SHEM (supplemented with 666 KIU/ml of aprotinin).

8) When the epithelium reached confluency (which usually takes 1 to 2weeks), air lift was performed (for about 1 week) The culture mediumused was SHEM without aprotinin.

9) The cultured epithelium was detached with a scraper.

A photographic image (×100) of the rabbit oral mucosal epithelial cellsgrown on the fibrin sheet observed under an optical microscope is shownin FIG. 19. A photographic image of the cultured epithelium sheetdetached with a scraper is shown in FIG. 20. Using rabbit oral mucosalepithelial cells cultured on a fibrin sheet, a thick epithelium sheetcould be prepared by means of co-cultivation of 3T3 cells and airlifting.

(3) Amex Embedding Fixation of Fibrin Sheet Culture

The epithelium sheet was embedded by the Amex method according to thefollowing procedure.

1) The epithelium sheet was placed in a net.

2) The sample sheet was immersed in acetone at 4° C.

3) The sheet was transferred along with acetone to −20° C. and fixed for24 hours.

4) The acetone was replaced by a fresh aliquot (4° C.) to impregnate thesheet for 15 min.

5) The acetone was replaced by a fresh aliquot (room temperature) toimpregnate the sheet for 15 min.

6) The acetone was replaced by methyl benzoate to impregnate the sheetfor 15 min. This step was repeated twice.

7) The methyl benzoate was replaced by xylene to impregnate the sheetfor 15 min. This step was repeated twice.

8) The sheet was impregnated with paraffin for 1 hour. This step wasrepeated three times.

9) The sheet was embedded in paraffin.

(4) Immunostaining of Fibrin Sheet Culture

With respect to each of the samples of rabbit corneal tissue, oralmucosal tissue, cultured corneal epithelium and cultured oral mucosalepithelium, a paraffin-embedded section was immunostained according tothe following procedure.

1) The section was treated with 3% H₂O₂ at room temperature for 30 min.

2) Blocking (10% normal donkey serum—1% BSA—0.01M PBS) of the sectionwas performed at room temperature for 1 hour.

3) The section was reacted with a primary antibody (AE5: PROGEN 61807:1/1000). Isotype control (mouse IgG1: Dako: 1/500) O/N.

4) The section was reacted with a secondary antibody (donkey anti-MsIgG-FITC, Jackson: 711-095-152: 1/50) at room temperature for 30 min.

5) Nuclear staining (DOJINDO: 1 μg/ml DAPI) was performed at roomtemperature for 5 min.

The immunostaining (AE5) of the rabbit corneal epithelial tissue andoral mucosal epithelial tissue is shown in FIG. 21. Blue: nuclearstaining (DAPI). Green: AE5 staining (keratin 3/12). The keratin 3/12 inthe epithelium of the cornea and the oral mucosa as positive controlsshowed staining of epithelial cells in both tissues (FIGS. 21B and D).

The immunostaining (AE5) of the rabbit cultured corneal epithelial cellsand cultured oral mucosal epithelial cells is shown in FIG. 22. In thecultured two types of epithelium, the corneal epithelium formed multiplelayers and the staining of keratin 3/12 was observed (FIGS. 22B and D).The oral mucosal epithelium also formed multiple layers, but thestaining of keratin 3/12 was observed weakly.

As mentioned above, rabbit corneal epithelium and oral mucosalepithelium cultured on fibrin sheets formed multiple layers andexpressed keratin. Accordingly, it was demonstrated that culturedepithelium having properties similar to those of normal tissue could beprepared.

All publications, patents and patent applications cited herein areincorporated herein by reference in their entirety.

INDUSTRIAL APPLICABILITY

Using the cell sheets manufactured by the method of the presentinvention, it becomes possible to create analysis models for variousconditions (e.g., arrhythmia in myocardial cells) and to transplanttissues at the clinical level, for example, in regenerative medicine. Inaddition, the cell sheets manufactured by the method of the presentinvention can be used to conduct various biological activity tests foragents, thus enabling screening for candidate substances as medicinesand agricultural chemicals having desired biological activities.

1-5. (canceled) 6: A composition for use in coating a substrate surfacewith fibrin for cell sheet preparation, the composition comprisingfibrinogen, thrombin and physiological saline, wherein the content offibrinogen is 45-180 mg per 16 ml of physiological saline and thecontent of thrombin is 0.2-0.8 U per 16 ml of physiological saline. 7: Amethod for manufacturing a substrate having a surface coated with fibrinfor cell sheet preparation, comprising coating the substrate surfacewith the composition according to claim
 6. 8: A method for manufacturinga cell sheet which does not substantially contain fibrin, comprisingculturing cells on a fibrin-coated surface of a substrate until thecells reach confluency; continuing the cultivation of the cells for asufficient time period to cause the degradation of fibrin at the bottomof the cells; and detaching the cultured cells from the substratesurface in a sheet-like form to give a cell sheet. 9: The methodaccording to claim 8, further comprising overlaying the detachedsheet-like cultured cells. 10: The method according to claim 8, whereinthe cell sheet is utilized in the field of regenerative medicine orutilized in a biological activity test of an agent. 11: A method formanufacturing a cell sheet which does not substantially contain fibrin,comprising culturing cells on a substrate surface coated with thecomposition according to claim 6 until the cells reach confluency;continuing the cultivation of the cells for a sufficient time period tocause the degradation of fibrin at the bottom of the cells; anddetaching the cultured cells from the substrate surface in a sheet-likeform to give a cell sheet. 12: The method according to claim 11, furthercomprising overlaying the detached sheet-like cultured cells. 13: Themethod according to claim 11, wherein the cell sheet is utilized in thefield of regenerative medicine or utilized in a biological activity testof an agent.